CHARACTERIZATION OF PROTEINS DURING AGGREGATION USING TURBIDIMETRY

Abstract

The dissolution process for hydrophobic proteins as well as protein denaturation-renaturation reactions often lead to the formation of aggregates. The characterization of the aggregates in terms of sire and the characterization of the physico-chemical changes undergone by the proteins are key to the elucidation of the kinetics of both, the aggregation processes and the denaturation-renaturation reactions. The aggregation processes and the denaturation-renaturation reactions are amenable to characterization through spectroscopy techniques. This paper reports on a technique for the estimation of the particle size and the absorption coefficients of proteins in solution and in protein aggregates present during denaturation-renaluration reactions. The technique reported is based on the application of Mie theory to turbidity measurements of polydisperse particulate systems. Mie theory is applied to both, the region where the particles are non-absorbing and, to the spectral region where the protein intrinsic chromophores absorb. This technique yields estimates of the scattering corrected absorption coefficients and of the relative refractive index of the aggregates. The results obtained indicate that subsequent application of spectral deconvolution techniques can yield quantitative information on the spectral changes undergone by the intrinsic protein chromophores upon denaturation or renaturation